FIELD OF THE INVENTION
[0001] The present invention relates to the use of neuregulin (NRG) for the preparation
of a drug for preventing, treating or relieving myocardial injury in mammals. The
present invention also relates to a method of administration, a frequency of administration
and an administered dose of the pharmaceutical preparation or composition containing
NRG for preventing, treating or relieving myocardial injury in mammals. In particular,
the present invention relates to a method for preventing, treating or relieving myocardial
injury and a pharmaceutical composition containing NRG therefor, and a method of administration,
a frequency of administration and an administered dose of the pharmaceutical preparation
or composition containing NRG for preventing, treating or relieving myocardial injury
in mammals.
BACKGROUND OF THE INVENTION
[0002] Cardiovascular problems pose a serious threat to the people's lives and health on
a global scale. There are many types of cardiovascular diseases, including heart failure,
myocardial infarction, coronary atherosclerotic heart disease, arrhythmia, cardiomyopathy,
valvular heart disease, infective endocarditis, pericardial disease, ischemic heart
disease, congenital heart disease, etc. A cardiovascular disease tends to cause myocardial
injury and affect the cardiac function, thereby making the human body unhealthy. Myocardial
infarction is a cardiovascular disease that seriously endangers the human health.
As the people's living conditions are continuously ameliorated, the incidence of ischemic
myocardial infarction increases steadily. Myocardial infarction is a type of ischemic
myocardial necrosis that is mostly induced by severe and chronic myocardial ischemia
caused by sharp reduction or interruption in coronary blood supply due to persistent
blockade in the coronary artery. Ischemic myocardial infarction causes cardiomyocyte
necrosis and scarring, thereby affecting the cardiac function.
[0003] When myocardial infarction occurs, the coronary artery is occluded for 20-30 min,
leading necrosis of some cardiomyocytes owing to blood insufficiency, starting the
pathological process of myocardial infarction. As myocardial necrosis becomes severer
and severer, most of the affected cardiomyocytes gradually suffer from coagulative
necrosis 2 hours later, while the myocardial interstitium is congestive and oedematous,
accompanied by abundant inflammatory cell infiltration. The myocardial necrosis process
almost comes to an end 6-12 hours later. The myocardial fibers dissolve 1-2 weeks
later, i.e., it is swallowed by macrophages, getting gradually fibrillated until the
necrotic zone is completely replaced by dense fibrous scars 6 weeks later. This is
known as old or healed myocardial infarction.
[0004] The normal function of the left ventricle is significantly affected after the occurrence
of myocardial infarction. If the heart suffers from ischemia on a large scale, the
pumping function of the left ventricle may be impaired, leading to a decrease in cardiac
output, stroke volume and blood pressure while an increase in end-systolic volume
and a rise in end-diastolic volume within several weeks after the infarction.
[0005] Ventricular remodeling is an important pathological behavior that occurs following
myocardial infarction. Post-infarction ventricular remodeling refers to a change that
takes place in the structure and morphology of the ventricular infarct area and non-infarct
area after the occurrence of myocardial infarction: The change in the infarct area
mainly includes infarct expansion while the change in the non-infarct area is primarily
manifested in ventricular enlargement. The typical manifestation of ventricular remodeling
is ventricular decompensated hypertrophy; a change in the ventricular mass, volume
gain and morphologic alteration can cause ventricular pumping dysfunction, which may
progress into heart failure. The basic mechanism that causes the occurrence and progression
of heart failure is ventricular remodeling. The symptom is a persistent and progressive
change second to myocardial infarction. Its severity determines the patient's cardiac
function and prognosis. Post-infarction ventricular remodeling is one of the major
cardiovascular diseases that affects the patient's cardiac function and endangers
the human life.
[0006] Currently, the major therapies for myocardial infarction include early-stage reperfusion
(including thrombolytic therapy and interventional therapy), angiotensin (ANG) II
receptor blocker, angiotensin converse enzyme (ACE) inhibitor, β-receptor blocker,
etc., which are effective in reducing the infarct size, reducing recurrent myocardial
ischemia, improving revascularization and suppressing excessive ventricular dilatation,
thereby decreasing the incidence of chronic heart failure.
[0007] The symptoms of myocardial infarction are closely related to the infarct size and
site and the conditions of coronary collateral vessels. The cardinal symptoms include
pain, fever, tachycardia, nausea, vomiting, hypotension, shock, arrhythmia, etc. The
main complications of myocardial infarction include papillary muscle dysfunction or
rupture, heart rupture, ventricular aneurysm, embolism, post-infarction syndrome (PMIS),
etc.
[0008] Most of the existing drugs or interventional therapies can only allay the symptoms
of myocardial infarction, and cannot repair cardiac tissue injury. For patients with
advanced myocardial infarction, although heart transplant, as the last treatment choice,
can improve the cardiac function and save dying patients, it can hardly have a widespread
application in clinical practice owing to the scarcity of donors, the complexity of
surgery, immunological rejection and high treatment costs.
[0009] To sum up, the myocardial injury caused by a cardiovascular disease causes a serious
harm to human health. Especially, as a fatal disease that seriously endangers human
health, myocardial infarction needs to be treated with a safer and more effective
drug in clinical practice.
[0010] Neuregulin (NG) or heregulin (HRG), which is a member of the EGF-like family, refers
to a group of growth differentiation factors (GDFs) structurally similar to one another,
including NRG1, NRG2, NRG3 and NRG4, as well as their isomers, which exert a series
of biological effects in stimulating breast cancer cell differentiation and lactoprotein
secretion (
Lessor T et al., JCell Biochem. 1998; 70 (4): 587-595); inducing the differentiation of neural crest cells into Schwann cells (
Topilko et al., Mol Cell Neurosci, 1996; 8 (2-3): 71-75); stimulating the synthesis of acetylcholine receptors in skeletal muscle cells (
Altiok N et al., EMBO.J. 1995; 14 (17): 4258-4266); promoting cardiocyte survival and DM synthesis (
Zhao YY et al. I Biol Cher. 1998; 273 (17): 10261-10269). An in-vivo study conducted in mouse embryos with severe NRG gene deficiency proved
the necessity of NRG for cardiac and nerve development.
[0011] The NRG receptors are members of the EF receptor family, including FR, ErbB2, ErbB
and ErbB4, which play an important role in cell growth, differentiation, survival,
etc. They are tyrosine kinase receptors, composed of the extracellular ligand-binding
domain, transmembrane domain and intracellular tyrosine kinase domain. When NRG binds
to the extracellular domain of ErbB3 or ErbB4, a conformational change takes place
in it, thereby causing the formation of ErbB3/ErbB4 or ErbB2/ErbB3 heterodimers or
ErbB4/ErbB4 homodimers, and causing the phosphorylation of its C-terminus. The phosphorylated
C-terminus can further bind to the downstream signaling proteins in the cells to activate
the AKT and/or EK signaling pathways, ultimately causing a series of cell responses
such as stimulation or inhibition of cell proliferation, cell differentiation, cell
apoptosis, cell migration or cell adhesion. Among these receptors, ErbB2 and ErhB4
are mainly expressed in the cardiac tissues (
Zhao YY et al., Circ Res. 1999; 84 (12): 1380-1387).
[0012] Existing evidence shows that the EGF-like domain of NRG-1, which contains 50-64 amino
acid, is highly capable of binding to and activating the receptors (
Culousoou JM et al., J Biol Chem. 1995; 270 (21): 12857-12863). NKG-1β can bind toErbB3 and ErbB4 with high affinity. ErbB2 can form a heterodimer
with ErbB3 or ErhB4, and its affinity for the ligand is higher than that of the ErbB3
or ErbB4 homodimer for the ligand. The neurodevelopmental studies confirm that the
formation of the sympathetic nervous system requires signal transduction via NRG-1β,
ErbB2 and ErhB3 (
Britsch S et al., Dienes Dev. 1998; 12 (12): 1825-1836). When the expression of NRG-1β or ErbB2 or ErbB4 is distributed, embryonic death
is caused by cardiac developmental defects (
Gassmann M et al., Nature, 1995; 378(6555): 390-394). Recent studies indicate that NRG-1β, ErbB2, and ErbB4 not only are essential for
cardiac development, but also play a very important role in maintaining the cardiac
function of adults (
Kuramochi Y et al., J Mol Cell Cardia1. 2006; 41 (2): 228-235). NRG-1β has been proved to be able to strengthen the formation of myocardial sarcomeres
in adults. It has been found in various animal models of heart failure that the intake
of MRG-1β EGF-like domain can ameliorate the cardiac function to prevent cardiac dysfunction
(
Liu et al., J Am Coll Cardiol. 2006; 48: 1438-1447). In clinical trials, NRG also showed a therapeutic effect on chronic heart failure
caused by various etiological factors, significantly enhancing the cardiac function
(
CN200910057390.5). In the animal model of cerebral ischemia-reperfusion, NRG-1 also showed a significant
protective effect on the brain cells, inhibiting brain cell apoptosis, enhancing the
neurologic function and reducing the infarct size (
Li Q et al., Neurosci Lett. 2008; 443 (3): 155-159). There is evidence that cardiac ischemia-reperfusion induces the release of NRG-1
and activates the NRG/ErbB signaling pathway in cardiocytes (
Kuramochi Y et al., J Biol Chem. 2004; 279 (49): 51141-51147), and that NRG-1 plays a role in preventing, treating or relieving cardiac ischemia-reperfusion
injury (
WO2011091723).
[0013] Myocardial injury is a fatal disease that severely endangers human health, but the
method of administration, frequency of administration and administered dose of NRG-1
for the treatment of myocardial injury have yet to be clearly determined. The present
invention provides a method and pharmaceutical composition containing NRG therefor
to meet the above needs. It is worth mentioning that the present invention particularly
provides an optimized frequency of administration, the present invention particularly
provides an optimized administered dose, and the present invention particularly provides
an optimal method of administration. The present invention further relates to the
use of NRG in the preparation of a drug for preventing, treating or relieving myocardial
injury in mammals. For the treatment of myocardial infarction, the present invention
particularly provides an optimized frequency of administration, the present invention
particularly provides an optimized administered dose, and the present invention particularly
provides an optimal method of administration.
DETAILED DESCRIPTION
A. SUMMARY
[0014] The present invention provides uses of NRG for the preparation of a drug for preventing,
treating or relieving myocardial injury in mammals. The present invention further
relates to the use of NRG for the preparation of a drug for preventing, treating or
relieving myocardial injury in mammals, and the mammals are preferably humans. NRG
can improve the cardiac function affected by myocardial injury and reduce cardiac
remodeling.
[0015] Many cardiovascular diseases, such as heart failure, myocardial infarction, coronary
atherosclerotic heart disease, arrhythmia, myocarditis, valvular heart disease, infective
endocarditis, pericardial disease, ischemic heart disease, congenital heart disease,
etc., can cause myocardial injury. Myocardial injury affects the cardiac function,
thus jeopardizing human health. Myocardial infarction is accompanied by prolonged
coronary occlusion, usually by cardiocyte apoptosis and necrosis, mass inflammatory
cell infiltration and myocardial fibrosis, which induce myocardial injury. Myocardial
infarction injury tends to cause cardiac dysfunction, thus affecting human health.
[0016] The present invention is based on a scientific discovery that NRG is critical to
cardiac development and also plays a very important role in maintaining the cardiac
function of adults; the present invention is based on a scientific discovery that
NRG can strengthen the formation of myocardial cell sarcomeres, cytoskeleton and intercellular
junction; the present invention is based on a scientific discovery that NRG can enhance
the cardiac function of the animals or patients with heart failure in various animal
models and clinical trials; the present invention is based on a scientific discovery
that NRG exerts a protective effect on brain cells in an animal model of cerebral
ischemia-reperfusion; the present invention is based on a scientific discovery that
NRG exerts a protective effect on brain cells in an animal model of cardiac ischemia-reperfusion;
NRG, NRG polypeptides and NRG mutants or other complexes with the NRG-like function
are all within the scope of the present invention.
[0017] In the first aspect, the prevention invention provides a pharmaceutical preparation
for preventing, treating or relieving myocardial injury in mammals. The prevention
invention further provides a pharmaceutical preparation for preventing, treating or
relieving myocardial infarction injury in mammals, and the mammals are preferably
humans. The pharmaceutical preparation contains an effective amount of NRG or its
functional fragments, or a nucleic acid encoding NRG or its functional fragments,
or a substance that increases the production and/or function of NRG, and a pharmaceutically
acceptable carrier, excipient, etc. The pharmaceutical preparation can be used in
combination with other drugs or therapies for preventing, treating or relieving myocardial
injury. In one embodiment, the pharmaceutical preparation containing NRG is effective
in increasing the EF value of the mammalian left ventricle. In another embodiment,
the pharmaceutical preparation containing NRG is effective in reducing the left ventricular
end-diastolic volume (LVEDV) or left ventricular end-systolic volume (LVESV). In another
embodiment, the pharmaceutical preparation containing NRG is subcutaneously injected
via a syringe or another device. In another embodiment, the pharmaceutical preparation
containing NRG is subcutaneously injected via a pump, such as an injection pump. In
some embodiments, the syringe pump is a micropump. In a further embodiment, the micropump
is an insulin pump. It is worth mentioning that the present invention is suitable
for any pharmaceutically available preparation, and the pharmaceutical preparation
contains the NRG described above or contains NRG and a pharmaceutically acceptable
excipient, diluent or carrier. The pharmaceutical preparation used in the present
invention includes but is not limited to the content of the present application.
[0018] In the second aspect, the present invention provides a method for preventing, treating
or relieving myocardial injury in mammals. The present invention further provides
a method for preventing, treating or relieving myocardial infarction injury in mammals,
and the mammals are preferably humans. The method comprises the use of an effective
amount of NRG or its functional fragments, or a nucleic acid encoding NRG or its functional
fragments, or a substance that increases the production and/or function of NR required
for preventing, treating or relieving myocardial injury in mammals. Other drugs can
be used in combination with the method, in particular an effective amount of NRG or
its functional fragments, or a nucleic acid encoding NRG or its functional fragments,
or a substance that increases the production and/or function of NR for preventing,
treating or relieving myocardial injury in mammals.
[0019] In the third aspect, the present invention provides a composition for preventing,
treating or relieving myocardial injury in mammals. The present invention further
provides a pharmaceutical composition for preventing, treating or relieving myocardial
infarction injury in mammals, and the mammals are preferably humans. The pharmaceutical
composition contains a type of NRG provided by the present invention for preventing,
treating or relieving myocardial injury in mammals, and other drugs for preventing,
treating or relieving myocardial injury. The pharmaceutical composition comprises
an EGF-like domain, and the domain has been proven to be able to bind to and activate
the receptors. In particular, as an example, but not for the purpose of limitation,
the NRG provided by the present invention is a fragment of the NRG-1β2 isomer, and
it comprises 177-237 amino acid. The amino acid sequence of the fragment is as follows:

[0020] In the fourth aspect, the present invention provides an administered dose of the
pharmaceutical preparation containing NRG used for preventing, treating or relieving
myocardial injury in mammals. The present invention further provides an administered
dose of the pharmaceutical preparation containing NRG used for preventing, treating
or relieving myocardial infarction injury in mammals, and the mammals are preferably
humans. The effective dose means that one or more beneficial effects can be achieved
when the dose of NRG is applied to a mammal. The beneficial effects can improve the
cardiac function of patients with myocardial injury, or prevent further deterioration
of their cardiac function, or suppress the deterioration of cardiac dysfunction that
may be caused by myocardial injury. The dose provided by the present invention for
a mammal is from 0.1 µg/kg/day (protein/body weight) to 360 µg/kg/day (protein/body
weight). In one embodiment, the dose is from 0.3 µ/kg/day (protein/body weight) to
50 µg/kg/day (protein/body weight); in one embodiment, the effective dose is 7.5 µg/kg/day;
in one embodiment, the effective dose is 15 µg/kg/day; in another embodiment, the
effective dose is 30 µg/kg/day.
[0021] In the fifth aspect, the present invention provides a method of administration of
the pharmaceutical preparation containing NRG for preventing, treating or relieving
myocardial injury in mammals. The present invention further provides a method of administration
of the pharmaceutical preparation containing NRG for preventing, treating or relieving
myocardial infarction injury in mammals, and the mammals are preferably humans. The
pharmaceutical preparation can be taken by oral administration, rectal administration,
topical administration, inhalation administration, buccal administration (e.g. sublingual
administration), parenteral administration (e.g. subcutaneous injection, intramuscular
injection, intracutaneous injection or intravenous injection), transdermal administration
or other proper methods. In one embodiment, NRG is administered only once a day. In
another embodiment, NRG is administered multiple times a day. In one embodiment, NRG
is administered in one day. In another embodiment, this tolerance dose of NRG is administered
within a few days. In another embodiment, NRG is administered multiple times a day,
for many consecutive days. In another embodiment, NRG is administered 2 days a week,
multiple times a day, for many consecutive weeks. In another embodiment, NRG is injected
subcutaneously 2 days a week, 3 times a day, for many consecutive weeks. In another
embodiment, NRG is injected subcutaneously 3 times a day, for many for consecutive
days. In another embodiment, NRG is injected subcutaneously 3 times a day, for 35
consecutive days. In another embodiment, NRG is injected subcutaneously 3 times a
day, for 38 consecutive days. In another embodiment, NRG is injected subcutaneously
3 times a day, for 49 consecutive days. In another embodiment, NRG is injected subcutaneously
3 times a day, for 60 consecutive days. In another embodiment, NRG is injected subcutaneously
3 times a day, for more than 35 consecutive days. In another embodiment, NRG is administered
multiple times a day, for many consecutive days, followed by slow withdrawal. In another
embodiment, NRG is administered multiple times a day, for many consecutive days, followed
by three-week slow withdrawal: administered every other day in the first week; administered
every three days in the second week; injected subcutaneously every four days in the
third week. In another embodiment, NRG is injected subcutaneously 3 times a day, for
more than 38 consecutive days, followed by slow withdrawal. In another embodiment,
NRG is injected subcutaneously 3 times a day, for 49 consecutive days, followed by
three-week slow withdrawal: administered every other day in the first week; administered
every three days in the second week; injected subcutaneously every four days in the
third week. In another embodiment, NRG is administered multiple times a day, for many
consecutive days, followed by slow reduction in daily dose. In another embodiment,
NRG is administered 3 times a day, for many consecutive days, followed by slow reduction
in daily dose. In another embodiment, NRG is s injected subcutaneously 3 times a day,
for more than 60 consecutive days, followed by slow reduction in daily dose. In another
embodiment, NRG is injected subcutaneously 3 times a day, for 60 continuous days,
followed by three-week slow withdrawal: the daily dose is half of the continuously
administered dose in the first week; the daily dose is a quarter of the continuously
administered dose in the second week; the daily dose is one eighth of the continuously
administered dose in the third week.
[0022] The present invention also provides a kit for preventing, treating or relieving myocardial
injury in mammals. The present invention also provides a kit for preventing, treating
or relieving myocardial infarction injury in mammals, and the mammals are preferably
humans. The kit contains a single dose or multiple doses of the aforesaid pharmaceutical
preparation or composition for preventing, treating or relieving myocardial injury,
and instructions on how to use the pharmaceutical preparation or composition.
[0023] The pharmaceutical preparation or composition provided by the present invention can
be administered before, during or after the occurrence of a cardiac disease. When
used for prevention, the pharmaceutical preparation or composition is generally administered
before the occurrence of the cardiac disease. When used for treatment, the pharmaceutical
preparation or composition is generally administered during or after the occurrence
of the cardiac disease. In one embodiment, the pharmaceutical preparation or composition
provided by the present invention is administered before the occurrence of the cardiac
disease. In another embodiment, the pharmaceutical preparation or composition provided
by the present invention is administered when myocardial infarction occurs. In another
embodiment, the pharmaceutical preparation or composition provided by the present
invention is administered after the occurrence of the cardiac disease.
[0024] The pharmaceutical preparation or composition provided by the present invention can
be taken by oral administration, rectal administration, topical administration, inhalation
administration, buccal administration (e.g. sublingual administration), parenteral
administration (e.g. subcutaneous injection, intramuscular injection, intracutaneous
injection or intravenous injection), transdermal administration or other proper methods.
Subcutaneous injection can be performed using a syringe, a pump (a microinfusion pump)
or another administration apparatus. The dosage forms of the pharmaceutical preparation
or composition provided by the present invention include but are not limited to tablets,
lozenges, cachets, dispersant, suspension liquid, solution, capsules, ointment and
similar forms.
B. DEFINITIONS
[0025] Unless otherwise defined, all the scientific and technical terms used herein have
the same meaning as is understood by those skilled in the art. All the patent documents,
patent application documents, published patent documents and other publications are
taken as reference. If any definition covered in the present section has a meaning
different from what is explained in the aforesaid documents, the explanation given
in the present section shall prevail.
[0026] Unless otherwise specified, "a/an", as used herein, means "at least one" or "one
or more than one".
[0027] "Mammals", as used herein, refer to non-human primates (bovines, pigs, horses, cats,
dogs, rats, mice, etc.) or primates (monkeys, humans), and are preferably humans.
[0028] "Myocardial injury", as used herein, refers to a type of myocardial damage caused
by a pathological cardiac disease such as heart failure, myocardial infarction, coronary
atherosclerotic heart disease, arrhythmia, myocardial disease, valvular heart disease,
infective endocarditis, pericardial disease, ischemic heart disease or congenital
heart disease. Myocardial injury tends to cause cardiac dysfunction, thereby affecting
human health. The pathogenesis of myocardial damage is related to multiple pathophysiological
changes, including the production of oxyradical, calcium overload, inflammatory reaction
caused by neutrophil invasion into the injured area, cardiocyte apoptosis or necrosis,
metabolic disorders of tissues caused by energy supply imbalance, abnormal cardiac
signal transduction, cholesterol accumulation, and formation of atherosclerotic plaques.
[0029] "Neuregulin" or "NRG", as used herein, refers to a protein or polypeptide that can
bind to and activate ErbB2, ErbB3, ErbB4 or a heterodimer or homodimer. It includes
the NRG isoforms, the EGF-like domain in NRG, the polypeptides containing the EGF-like
domain of NRG, NRG mutants or derivatives, and other gene products of NRG that are
capable of activating the above-mentioned receptors. NRG also includes NRG-1, NRG-2,
NRG-3 and NRG-4, polypeptides, fragments, and complexes with the NRG-like function.
Preferably, NRG is a type of protein or polypeptide that can bind to and activate
ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers. As an example, but not for the purpose of
limitation, the NRG (rhNRG) provided by the present invention is a fragment of the
NRG-1β2 isomer, i.e., 177-237 amino acid fragment, which contains an EGF-like domain.
The amino acid sequence of the fragment is as follows: SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMA
SFYKAEELYQ (SEQ ID NO: 1). The NRG used in the present invention can activate the
aforesaid receptors and regulate their biological functions, e.g. it can stimulate
skeletal muscle cells to synthesize an acetylcholine receptor; promote the differentiation,
survival and DNA synthesis of cardiocytes. NRG also comprises the conservative NRG
mutants that do not substantially affect its biological functions. As is clear to
those skilled in the art, the mutation of a single amino acid in a non-critical zone
does not cause a change in the biological functions of the protein or polypeptid (
Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin /Cummings
Pub. co., p.224). The NRG used in the present invention can be extracted from natural resources or
obtained by recombinant technology, artificial synthesis or other means.
[0030] "EGF-like domain", as used herein, refers to polypeptide fragment encoded by the
NRG gene that can bind to and activate ErbB2, ErbB3, ErbB4 or its heterodimer or homodimer,
and has a structure similar to the EGF receptor binding zone described in the literature
below:
WO 00/64400;
Holmes et al., Science, 256: 1205-1210 (1992);
U.S. Patent Nos. 5,530,109 and
5,716,930;
Hijazi et al., Int. J. Oncol., 13: 1061-1067 (1998);
Chang et al., Nature, 387: 509-512 (1997);
Carraway et al., Nature, 387: 512-516 (1997);
Higashiyama et al., J. Biochem., 122: 675-680 (1997); and
WO 97/ 09425. In some embodiments, the EGF-like domain binds to and activates ErbB2/ErbB4 or ErbB2/ErbB3
heterodimers. In some embodiments, the EGF-like domain comprises the amino acids in
the receptor binding zone of NRG-1. In some embodiments, the EGFP-like domain refers
to the 177-226, 177-237 or 177-240 amino acid of NRG-1. In some embodiments, the EGFP-like
domain comprises the amino acids in the receptor binding zone of NRG-2. In some embodiments,
the EGF-like domain comprises the amino acids in the receptor binding zone of NRG-3.
In some embodiments, the EGF-like domain comprises the amino acids in the receptor
binding zone of NRG-4. In some embodiments, the EGF-like domain comprises the amino
acid sequence described in
US Patent 5,834,229: AlaGluLysGlu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser
Asn Pro.
[0031] NRG can be made into an agent that can be taken by oral administration, rectal administration,
topical administration, inhalation administration, buccal administration (e.g. sublingual
administration), parenteral administration (e.g. subcutaneous injection, intramuscular
injection, intracutaneous injection or intravenous injection), transdermal administration
or other proper methods. In all modes of administration, the most suitable route of
administration needs to be selected according to the treatment conditions and severity,
as well as the properties of the specific NRG used. NRG can be administered alone.
Or more suitably, NRG can be administered with some pharmaceutically acceptable carriers
or excipients. Any suitable, pharmaceutically acceptable carrier or excipient applies
to the current method (
Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack
Publishing Company, April 1997).
[0032] The "pump" used herein is an administration apparatus for subcutaneous injection
of a pharmaceutical liquid, drug, protein and/or other compositions. It can be used
for continuous, accurate and quantitative administration. The pump is provide with
a subcutaneous catheter for continuous subcutaneous infusion. The catheter can be
placed externally or the catheter port can be embedded in the pump mechanism. The
microinfusion pump is portable and easy-to-use device that can be used for accurate
infusion. For example, an insulin pump is a medical device used to administer insulin
or other drugs during the treatment of diabetes or other diseases. It is also considered
to be used for continuous subcutaneous insulin delivery. The insulin pump can be equipped
with a disposable thin-walled plastic pipe or a catheter so that insulin or other
drugs can enter the tissues. The catheter can be inserted subcutaneously and translocated
as needed. The pump can be mounted in an external device that can be connected to
the patient, or in a device that can be implanted in the patient's body. An external
pump refers to a device designed for use in a fixed location such as a hospital, a
clinic or a similar place, in particular referring to a mobile or portable device,
such as a pump that can be carried by a patient or a similar device. The external
pump comprises reservoir capable of storing a fluid medium, such as but not limited
to a fluid medium containing NRG.
[0033] The external pump can be connected to the patient through flowing liquid, e.g. through
a proper hollow tube. The hollow tube can be connected with a hollow needle, which
is used to pierce the patient's skin for infusion. Alternatively, the hollow tube
can be directly connected to the patient through a cannula or a similar object. The
external pump can be worn by the patient or attached to or underneath the patient's
clothes. An appropriate pump refers to but is not limited to a microinfusion pump
that can be used for high-frequency infusion, such as the MiniMedParadigm522 insulin
pump, MiniMedParadigm722 insulin pump, MiniMedParadigm515 insulin pump, MiniMedParadigm715
insulin pump, MiniMedParadigm512R insulin pump, MiniMed Paradigm712R insulin pump,
MiniMedParadigm508 insulin pump, and MiniMedParadigm508R insulin pump (Medtronic,
Northridge, Canada), and other similar devices well-known to those skilled in the
art.
[0035] As use herein, "other drugs or therapies that can be used to prevent, treat or relieve
myocardial injury" refer to the drugs and interventional therapies that are proverbially
applicable to the treatment of myocardial injury, as well as the drugs and interventional
therapies that are proverbially applicable to the treatment of myocardial infarction
injury. Among them, the drugs for treating myocardial infarction include antiplatelet
drugs (aspirin, clopidogrel, etc.), anticoagulants (heparin, bivalirudin, etc.), thrombolytic
agents (alteplase, tenecteplase, urokinase, recombinant human pro-urokinase, etc.),
lipid-lowering agents (statins, cholesterol absorption inhibitors), angiotensin converse
enzyme inhibitors/ANG II receptor blockers, β receptor blockers, calcium channel blockers,
nitric acid esters, phosphatase inhibitors, diuretics, renin-angiotensin-aldosterone
system (RAS) antagonists, myocardial energy optimizers, drugs for improving ischemic
tissue metabolism, free radical scavengers, etc. The interventional therapies include
coronary interventional therapy, etc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036]
Figure 1 shows the echocardiographic results of the effect of NRG in treating rats
with acute myocardial infarction through long-term subcutaneous administration of
different doses
Figure 2 shows the echocardiographic results of the effect of NRG in treating rats
with acute myocardial infarction through long-term subcutaneous administration at
different frequencies
Figure 3 shows the therapeutic effect of NRG on acute myocardial infarction in rats
through long-term subcutaneous administration followed by withdrawal through frequency
reduction
Figure 4 shows the therapeutic effect of NRG on acute myocardial infarction in rats
through long-term subcutaneous administration followed by withdrawal through dose
reduction
EXAMPLES
Example 1: Therapeutic effect of rhNRG on acute myocardial infarction in rats through
long-term subcutaneous administration of different doses - A study on the dose-effect
relationship of NRG
1. Objective
[0037] To investigate the dose-effect relationship of NRG for treatment of acute myocardial
infarction in rats by observing the therapeutic effect of different doses of rhNRG
on acute myocardial infarction in rats in a rat model of myocardial infarction caused
by left coronary artery ligation.
2. Experimental Drugs
[0038]
2.1 Excipient: developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
2.2 rhNRG: developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
3. Experimental Animals
[0039]
3.1 Strain and source: Wistar rats, provided by Shanghai Sippe-Bk Lab Animal Co.,
Ltd.
3.2 Gender, body weight and certificate: male, 200-270 g
4. Experimental Materials and Equipment
[0040]
An anesthesia apparatus, an isoflurane evaporator, from MSS INTERNATIONAL LTD.
Isoflurane 100 ml/bottle, from RWD Life Technologies Co., Ltd.
A cardiac ultrasonic detector Vivid E95
Ningbo Lingqiao suture needle with threads, from Ningbo Medical Needle Co., Ltd.
5. Experimental Methods
5.1 Establishment of a rat model of heart failure caused by coronary artery ligation
[0041] The rats were anesthetized with isoflurane through a gaseous anesthesia apparatus.
Then, the rats were fixed in a supine position. After chest hair removal, the skin
was disinfected with 75% alcohol. After the incision of the left anterior chest skin,
the thoracic muscles were bluntly separated, with the 4
th and 5
th ribs exposed. Hemostatic forceps were used to bluntly cut off the muscle between
the 4
th and 5
th ribs. Both hands were used to squeeze the heart out of the thoracic cavity so that
the heart should be fully exposed for observation of lung inflation and heartbeats.
The left atrial appendage and pulmonary conus were fully exposed to ligate the left
anterior descending coronary artery (LADCA) with surgical suture between them. The
heart was quickly restored upon after ligation. Then, the thoracic muscles and skin
were sutured. After surgery, the rats were put back into the cages for feeding and
close observations.
5.2 Grouping and administration
[0042]
Table 1 Experimental animal grouping and administration schedule
Group |
Administered Dose |
Route of Administration |
Frequency and Cyc e of Administration |
Control Group |
-- |
Subcutaneous injection |
3 times a day × 60 days |
High-dose NRG |
15 µg/kg/D |
Subcutaneous injection |
3 times a day × 60 days |
Middle-dose NRG |
7.5 µg/kg/D |
Subcutaneous injection |
3 times a day × 60 days |
Low-dose NRG |
3.75 µg/kg/D |
Subcutaneous injection |
3 times a day × 60 days |
[0043] Administration began on the day following the animal model establishment of myocardial
infarction.
5.3 Observation indexes
[0044] After being anesthetized with 4% isoflurane, the rats were fixed onto the operating
board in the left lateral recumbent position. The head of the rats was fixed in the
breathing mask of the gaseous anesthesia machine, with isoflurane used for maintenance
of anesthesia. After chest hair removal, the skin was disinfected with 75% alcohol
and coated with a coupling agent. An echocardiography probe was used to detect any
echo signal from the left ventricle of the rats. The left ventricular end-diastolic
diameter and left ventricular end-systolic diameter (D) were measured. The left ventricular
end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated. Also, the
ejection fraction (EF) value was found, EF = (EDV-ESV)/EDV*100%.
5.3.2 Data processing
[0045] All experimental data were expressed in ±SD.
6. Experimental Results
6.1 Echocardiographic results
[0046] Echocardiography was performed after 60 days of continuous NRG administration. The
results showed that the LVEDd, LVEDs and EF value of the excipient group were 0.971±0.07
cm, 0.832±0.08 cm and 34.6±7.00%, respectively; the LVEDd, LVEDs and EF value of the
15 µg/kg-NRG group were 0.975±0.07 cm, 0.794±0.10 cm and 42.9±11.32%, respectively;
the LVEDd, LVEDs and EF value of the 7.5 µg/kg-NRG group were 0.965±0.07 cm, 0.808±0.11
cm and 38.4±12.17%, respectively; the LVEDd, LVEDs and EF value of the 3.75 µg/kg-NRG
group were 0.994±0.08 cm, 0.839±0.12 cm and 37.0±12.23%, respectively. According to
the data of LVEDd and LVEDs, the LVEDd and LVEDs of the high-dose NRG group could
be reduced. According to the data of EF value, the cardiac function of the rats in
the high-dose, middle-dose and low-dose groups was all improved after 60 days of continuous
administration, and there was dose-effect relationship among the three groups. See
Table 2 and Figure 1 for details.
Table 2 Echocardiographic results of the effect of NRG in treating rats with acute myocardial
infarction through 60-day subcutaneous administration of different doses (
x±SD)
Time |
Dose (µg/kg) |
Group |
Number of animals |
LVEDd (cm) |
LVEDs (cm) |
EF (%) |
|
-- |
Excipient |
15 |
0.903±0.06 |
0.762±0.08 |
37.1±8.27 |
|
15 |
NRG |
18 |
0.908±0.05 |
0.759±0.08 |
40.6±9.15 |
DAY20 |
7.5 |
NRG |
16 |
0.924±0.05 |
0.756±0.08 |
411.9±15.17 |
|
3.75 |
NRG |
13 |
0.937±0.06 |
0.777±0.08 |
39.7±10.84 |
|
|
|
|
|
|
|
|
-- |
Excipient |
15 |
0.953±0.06 |
0.807±0.09 |
36.4±10.24 |
|
15 |
NRG |
18 |
0.946±0.08 |
0.775±0.11 |
42.2±11.27 |
DAY40 |
7.5 |
NRG |
16 |
0.946±0.08 |
0.782±0.12 |
40.5±13.39 |
|
3.75 |
NRG |
13 |
0.982±0.09 |
0.813±0.13 |
40.4±12.86 |
|
|
|
|
|
|
|
|
-- |
Excipient |
15 |
0.971±0.07 |
0.832±0.08 |
34.6±7.00 |
|
15 |
NRG |
18 |
0.975±0.07 |
0.794±0.10 |
42.9±11.32 |
DAY60 |
7.5 |
NRG |
16 |
0.965±0.07 |
0.808±0.11 |
38.4±12.17 |
|
3.75 |
NRG |
13 |
0.994±0.08 |
0.839±0.12 |
37.0±12.23 |
7. Conclusions
[0047] After 60-day treatment with rhNRG, the EF value of the treatment groups, which were
subcutaneously injected with 5 µg/kg, 2.5 µg/kg, 1.25 µg/kg of NRG 3 times a day,
was higher than that of the control group, and there was a certain dose-effect relationship
for the three doses.
Example 2: Therapeutic effect of rhNRG on acute myocardial infarction in rats through
long-term subcutaneous administration at different frequencies
1. Objective
[0048] To investigate the therapeutic effect of a certain dose of rhNRG on cute myocardial
infarction in rats through long-term subcutaneous administration at different frequencies
in a rat model of myocardial infarction caused by left coronary artery ligation.
2. Experimental Drugs
[0049]
2.1 Excipient: developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
2.2 rhNRG: developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
3. Experimental Animals
[0050]
3.1 Strain and source: Wistar rats, provided by Shanghai Sippe-Bk Lab Animal Co.,
Ltd.
3.2 Gender, body weight and certificate: male, 200-270g
4. Experimental Materials and Equipment
[0051] The same as "4. Experimental Materials and Equipment" in Example 1.
5. Experimental Methods
5.1 Establishment of a rat model of heart failure caused by coronary artery ligation
[0052] Established in the same way as the rat model of heart failure caused by coronary
artery ligation in 5.1, Embodiment 1
5.2 Grouping and administration
[0053]
Table 3 Experimental animal grouping and administration schedule
Group |
Administered Dose |
Route of Administration |
Frequency and Cycle of Administration |
Control Group |
-- |
Subcutaneous injection |
3 times a day × 35 days |
NRG/30µg/kg/Day |
30µg/kg/Day |
Subcutaneous injection |
3 times a day × 35 days |
NRG/30µg/kg/BIW |
30µg/kg/Day |
Subcutaneous injection |
3 times a day, 2 days a week × 5 weeks |
Nrg/30µG/KG/Day*7+QW |
30µg/kg/Day +30µg/kg/7 days |
Subcutaneous injection |
3 times a day × 7 days |
once a day, 1 day/week ×4 weeks |
[0054] All the experimental animals were randomly divided into groups upon coronary artery
ligation. According to post-ligation survival, the rats were randomly divided into
4 groups by body weight: the excipient group (control group), the NRG30µg/kgDay group,
the NRG30µg/kg/BlW group and the NRG30µgkgDay*7+QW group. Administration began on
the day following the the animal model establishment of myocardial infarction. For
the first three groups and for the fourth group on the first 7 days, the rats were
given subcutaneous injection 3 times a day and weighed once a day. They took medicine
by weight and the dose was 30µg/kg/day. For the fourth group in the last four weeks,
the rats were injected with NRG on one day per week, and the daily dose was 30µg/kg.
5.3 Observation indexes
5.3.1 Cardiac function test
[0055] After being anesthetized with 4% isoflurane, the rats were fixed onto the operating
board in the left lateral recumbent position. The head of the rats was fixed in the
breathing mask of the gaseous anesthesia machine, with isoflurane used for maintenance
of anesthesia. After chest hair removal, the skin was disinfected with 75% alcohol
and coated with a coupling agent. An echocardiography probe was used to detect any
echo signal from the left ventricle of the rats. The left ventricular end-diastolic
diameter and left ventricular end-systolic diameter (D) were measured. The left ventricular
end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated. Also, the
ejection fraction (EF) value was found, EF-(EDV-ESV)/EDV*100%. The cardiac function
of the rats was performed by echocardiography in the 1
st, 2
nd, 3
rd and 5
th weeks after the occurrence of myocardial infarction.
5.3.2 Data processing
[0056] All experimental data were expressed in ±SD.
6. Experimental Results
6.1 Echocardiographic results
[0057] Echocardiography was performed after 35 consecutive days of NRG administration. The
LVEDd, LVEDs, EF value of the control group were 0.925±0.084cm, 0.756±0.107cm and
42.5 ± 10.174%, respectively; the LVEDd, LVEDs, EF value of the NRG/30µg/kg/Day group
were 0.879 ± 0.058cm, 0.694 ± 0.077cm and 47.9 ± 8.342%, respectively; the LVEDd,
LVEDs, EF value of the NRG/30µg/kg/BIW group were 0.928±0.084cm, 0.746+0.110cm and
45.2+10.248%, respectively; the LVEDd, LVEDs, EF value of the NRG/30µg/kg/Day*7+QW
group were .931±0.070cm, 0.760±0.097cm and 42.7±9.892%, respectively.
[0058] After 35 days of continuous administration, as shown by the data of LVEDd and LVEDs,
NRG/30µg/kg/Day could significantly reduce the LVEDd and LVEDs; according to the data
of EF value, it was significantly higher in the NRG/30µg/kg/Day group than in the
control group; it showed a rising trend in the NRG/30µg/kg/BIW group compared with
the control group; compared with the control group, the cardiac function of the rats
in the NRG/30µg/kg/Day*7+QW group was improved to some extent on the first 7 days
of continuous administration, i.e., it showed a rising trend. Then, an injection was
given every 7 days to maintain the efficacy. See Table 4 and Figure 2 for the results.
Table 4 Echocardiographic results of the therapeutic effect of NRG on myocardial infarction
in rats through long-term subcutaneous administration at different frequencies (
x±SD)
Time |
Group |
Number of animals |
LVEDd (cm) |
LVEDs (cm) |
EF (%) |
|
Control Group |
52 |
0.800±0.060 |
0.629±0.074 |
48.6±7.992 |
|
NRG/30µg/kg/Day |
48 |
0.785±0.046 |
0.601±0.063 |
52.2±7.802 |
DAY8 |
NRG/30µg/kg BIW |
48 |
0.809±0.045 |
0.625±0.068 |
50.9±8.958 |
|
NRG/30µg/kg Day*7+QW |
50 |
0.785±0.045 |
0.607±0.061 |
50.9±7.337 |
|
Control Group |
50 |
0.863±0.058 |
0.696±0.081 |
44.6±9.297 |
|
NRG/30µg/kg/Day |
47 |
0.832±0.054 |
0.646±0.075 |
50.3±8.49 |
DAY 15 |
NRG/30µg/kg BIW |
48 |
0.856±0.065 |
0.671±0.091 |
48.9±9.893 |
|
NRG/30µg/kg Day*7+QW |
50 |
0.864±0.054 |
0.686±0.074 |
47.1±8.344 |
|
Control Group |
50 |
0.898±0.070 |
0.728±0.090 |
43.8±8.854 |
|
NRG/30µg/kg/Day |
46 |
0.859±0.057 |
0.670±0.069 |
49.5±7.897 |
DAY22 |
NRG/30µg/kg BIW |
48 |
0.896±0.074 |
0.711±0.102 |
47.1±10.427 |
|
NRG/30µg/kg Day*7+QW |
50 |
0.905±0.062 |
0.729±0.08 |
44.7±8.434 |
|
Control Group |
50 |
0.925±0.084 |
0.756±0.107 |
42.5±10.174 |
DAY36 |
NRG/30µg/kg/Day |
46 |
0.879±0.058 |
0.694±0.077 |
47.9±8.342 |
|
NRG/30µg/kg BIW |
48 |
0.928±0.084 |
0.746±0.110 |
45.2±10.248 |
|
NRG/30µg/kg Day*7+QW |
50 |
0.931±0.070 |
0.76±0.097 |
42.7±9.892 |
Example 3: Therapeutic effect of rhNRG on acute myocardial infarction in rats through
long-term subcutaneous administration followed by withdrawal through frequency reduction
1. Objective
[0059] To observe the therapeutic effect of rhNRG on acute myocardial infarction in rats
through long-term administration followed by withdrawal through frequency reduction
in a rat model of myocardial infarction caused by left coronary artery ligation.
2. Experimental Drugs
[0060]
2.1 Excipient: developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
2.2 rhNRG: developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
3. Experimental Animals
[0061]
3.1 Strain and source: Wistar rats, provided by Shanghai Sippe-Bk Lab Animal Co.,
Ltd.
3.2 Gender, body weight and certificate: male, 200-270g
4. Experimental Materials and Equipment
[0062] The same as "4. Experimental Materials and Equipment" in Embodiment 1
5. Experimental Methods
5.1 Establishment of a rat model of heart failure caused by coronary artery ligation
[0063] Established in the same way as the rat model of heart failure caused by coronary
artery ligation in 5.1, Embodiment 1
5.2 Grouping and administration
[0064] The rats were randomly divided into groups and medicated upon coronary artery ligation.
According to post-ligation survival, the rats were randomly divided into two groups,
including the excipient group and the NRG30 µg/kg group. There were 19 rats in the
excipient group and there were 18 rats in the NRG group. Continuous administration
of medicine started on the day following the modeling, with subcutaneous administration
for 3 times a day, at a dose of 10 µg/kg. Echocardiography was performed on day 14
after modeling. All the animals were medicated continuously until day 38, and the
animals in the NRG group were examined by echocardiography. The animals in the NRG
group were averagely divided into two subgroups, with the animals in one subgroup
continuing to be medicated while the animals in the other subgroup stopped taking
medicine at an early stage. The excipient group continued to be medicated. For the
continuous NRG administration subgroup, a 3-week withdrawal plan was implemented on
day 49: medicated every other day in the first week; medicated every three days in
the second week; subcutaneously injected with NRG every four days in the third week.
In terms of administration method, the rats were subcutaneously injected with NRG
3 times a day, exactly the same as above. For the NRG withdrawal subgroup, the clinical
symptoms of the rats were observed. All the animals underwent echocardiography every
week for monitoring of the changes in the cardiac function.
5.3 Observation indexes
5.3.1 Cardiac function test
[0065] After being anesthetized with 4% isoflurane, the rats were fixed onto the operating
board in the left lateral recumbent position. The head of the rats was fixed in the
breathing mask of the gaseous anesthesia machine, with isoflurane used for maintenance
of anesthesia. After chest hair removal, the skin was disinfected with 75% alcohol
and coated with a coupling agent. An echocardiography probe was used to detect any
echo signal from the left ventricle of the rats. The left ventricular end-diastolic
diameter and left ventricular end-systolic diameter (D) were measured. The left ventricular
end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated. Also, the
ejection fraction (EF) value was found, EF=(EDV-ESV)/EDV*100%.
5.3.2 Data processing
[0066] All experimental data were expressed in ±SD. GraphPad Prism6 was used for one-way
ANOVA analysis. P<0.05 indicates a significant inter-group difference; P<0.01 indicates
an extremely significant inter-group difference.
6. Experimental Results
6.1 Echocardiographic results
[0067] Echocardiography was performed after 35 consecutive days of NRG administration. The
LVEDd, LVEDs and EF value of the excipient group were 0.988±0.08 cm, 0.850±0.10 cm
and 33.6±11.36%, respectively; the LVEDd, LVEDs and EF value of the NRG group were
0.953±0.05 cm, 0.767±0.06 cm and 44.9±6.09%, respectively; the results showed that
NRG could significantly reduce the LVEDd and LVEDs and strengthen the cardiac systolic
function, thereby reversing left ventricular remodeling; after 49 days of continuous
administration, the LVEDd, LVEDs and EF value of the excipient group were 1.020±0.10
cm, 0.881±0.15 cm and 33.1±14.55%, respectively; the LVEDd, LVEDs and EF value of
the NRG withdrawal subgroup were 0.987± 0.05 cm, 0.807±0.06 cm and 42.2±5.48%, respectively,
while the LVEDd, LVEDs and EF value of the continuous NRG administration subgroup
were 0.973±0.07 cm, 0.783±0.08 cm and 45.0±5.51%, respectively; the results showed
that the sudden withdrawal of NRG exerted some effect on the cardiac function of the
rats. A gradual withdrawal plan was implemented in the continuous NRG administration
subgroup, and echocardiography was performed in the second week after drug withdrawal.
The LVEDd, LVEDs and EF value of the early NRG withdrawal subgroup were 1.043±0.06
cm, 0.887±0.06 and 35.4±6.78%, respectively; the LVEDd, LVEDs and EF value of the
gradual NRG withdrawal subgroup were 0.989±0.07 cm, 0.814±0.08 and 41.3±4.92%, respectively.
There was a significant difference from the excipient group. Echocardiography was
performed in the third week after withdrawal. The LVEDd, LVEDs and EF value of the
early NRG withdrawal subgroup were 1.010±0.06 cm, 0.842+0.06 cm and 38.9±5.04%, respectively;
the LVEDd, LVEDs and EF value of the gradual NRG withdrawal subgroup were 0.976±0.06
cm, 0.805±0.07 cm, 40.8±4.67%, respectively. Compared with the excipient group, the
effect of gradual withdrawal of NRG on the cardiac function of the rats was allayed.
See Tables 5 & 6 and Figure 3 for the results.
Table 5 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction
through 35-day subcutaneous administration (
x±SD)
Time |
Dose (µg/kg) |
Group |
Number of animals |
LVEDd (cm) |
LVEDs (cm) |
EF (%) |
DAY14 |
-- |
Excipient |
16 |
0.878±0.05 |
0.740±0.08 |
37.1±11.11 |
|
30 |
NRG |
18 |
0.860±0.04 |
0.694±0.06 |
44.5±7.85* |
DAY21 |
-- |
Excipient |
16 |
0.941±0.06 |
0.801±0.09 |
35.4±11.91 |
|
30 |
NRG |
18 |
0.905±0.04 |
0.725±0.06* |
45.5±7.96** |
DAY28 |
-- |
Excipient |
16 |
0.961±0.97 |
0.832±0.11 |
32.4±13.34 |
|
30 |
NRG |
18 |
0.928±0.05 |
0.749±0.06** |
44.3±6.95*** |
DAY35 |
-- |
Excipient |
16 |
0.988±0.08 |
0.850±0.10 |
33.6±11.36 |
|
30 |
NRG |
18 |
0.953±0.05 |
0.767±0.06** |
44.9±6.09*** |
***: p<0.001 for the subgroups after treatment compared with the excipient group;
**: p<0.01 for the subgroups after treatment compared with the excipient group;
*: p<0.05 for the subgroups after treatment compared with the excipient group |
Table 6 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction
in the two subgroups after 38-day subcutaneous administration (
x±SD)
Time |
Dose (µg/kg) |
Group |
Number of animals |
LVEDd (cm) |
LVEDs (cm) |
EF (%) |
DAY38 |
30 |
Administration |
9 |
0.953±0.06 |
0.764±0.07 |
44.9±7.03 |
|
-- |
Withdrawal |
9 |
0.952±0.05 |
0.767±0.07 |
44.7±6.26 |
|
-- |
Excipient |
16 |
1.005±0.09 |
0.883±0.11 |
29.8±11.43 |
DAY42 |
30 |
Administration |
8 |
0.948±0.04 |
0.755±0.04* |
46.5±4.01*** |
|
-- |
Withdrawal |
9 |
0.969±0.04 |
0.808±0.05 |
39.1±7.11* |
|
-- |
Excipient |
16 |
1.020±0.10 |
0.881±0.15 |
33.1±14.55 |
DAY49 |
30 |
Administration |
8 |
0.973±0.07 |
0.783±0.08* |
45.0±5.51** |
|
-- |
Withdrawal |
9 |
0.987±0.05 |
0.807±0.06 |
42.2±5.48* |
|
-- |
Excipient |
16 |
1.046±0.08 |
0.920±0.11 |
29.4±10.34 |
DAY56 |
30 |
Administration |
8 |
1.008±0.06 |
0.843±0.06* |
38.8±3.78* |
|
-- |
Withdrawal |
9 |
1.031±0.05 |
0.859±0.07 |
39.0±6.24* |
|
-- |
Excipient |
16 |
1.040±0.11 |
0.907±0.14 |
31.2±12.43 |
DAY63 |
30 |
Administration |
8 |
0.989±0.07 |
0.814±0.08* |
41.3±4.92* |
|
-- |
Withdrawal |
9 |
1.043±0.06 |
0.887±0.06 |
35.4±6.78 |
|
-- |
Excipient |
16 |
1.036±0.10 |
0.905±0.12 |
30.9±9.23 |
DAY68 |
30 |
Administration |
8 |
0.976±0.06 |
0.805±0.07 |
40.8±4.67* |
|
-- |
Withdrawal |
9 |
1.010±0.06 |
0.842±0.06 |
38.9±5.04* |
|
-- |
Excipient |
16 |
1.043±0.11 |
0.920±0.12 |
29.1±9.63 |
DAY74 |
30 |
Administration |
8 |
1.004±0.05 |
0.842±0.05* |
38.0±3.81* |
|
-- |
Withdrawal |
9 |
1.060±0.05 |
0.905±0.05 |
34.6±7.55 |
***: p<0.001 for the subgroups after treatment compared with the excipient group;
**: p<0.01 for the subgroups after treatment compared with the excipient group;
*: p<0.05 for the subgroups after treatment compared with the excipient group |
7. Conclusions
[0068] Through long-term subcutaneous administration followed by withdrawal through frequency
reduction, rhNRG can improve the cardiac function of the rats with myocardial infarction
and reduce cardiac remodeling.
Example 4: Therapeutic effect of rhNRG on acute myocardial infarction in rats through
long-term subcutaneous administration followed by withdrawal through dose reduction
1. Objective
[0069] To observe the therapeutic effect of rhNRG on acute myocardial infarction in rats
through long-term administration followed by withdrawal through frequency reduction
in a rat model of myocardial infarction caused by left coronary artery ligation.
2. Experimental Drugs
[0070]
2.1 Excipient: developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
2.2 rhNRG: developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
3. Experimental Animals
[0071]
3.1 Strain and source: Wistar rats, provided by Shanghai Sippe-Bk Lab Animal Co.,
Ltd.
3.2 Gender, body weight and certificate: male, 200-270g
4. Experimental Materials and Equipment
[0072] The same as "4. Experimental Materials and Equipment" in Embodiment 1
5. Experimental Methods
5.1 Establishment of a rat model of heart failure caused by coronary artery ligation
[0073] Established in the same way as the rat model of heart failure caused by coronary
artery ligation in 5.1, Embodiment 1
5.2 Grouping and administration
5.3 Observation indexes
[0074] The rats were randomly divided into groups and medicated upon coronary artery ligation.
According to post-ligation survival, the rats were randomly divided into 2 groups
by body weight. Subcutaneous injection was given 3 times a day, and the animals were
weighed once a day. The animals were medicated by weight. Echocardiography was performed
on day 10 after modeling. All the animals underwent echocardiography every 10 days,
underwent echocardiography every week after dose reduction, and underwent echocardiography
every 2 weeks after complete withdrawal. All the animals were medicated continuously
till day 60, and then a three-week dose-reduction withdrawal plan was implemented:
The administered dose was reduced to 15 µg/kg, 7.5 µg/kg and 3.75 µg/kg in the first,
second and third week, respectively. The drug was completely withdrawn after three-week
dose reduction to observe the clinical symptoms.
5.3.1 Cardiac function test
[0075] After being anesthetized with 4% isoflurane, the rats were fixed onto the operating
board in the left lateral recumbent position. The head of the rats was fixed in the
breathing mask of the gaseous anesthesia machine, with isoflurane used for maintenance
of anesthesia. After chest hair removal, the skin was disinfected with 75% alcohol
and coated with a coupling agent. An echocardiography probe was used to detect any
echo signal from the left ventricle of the rats. The left ventricular end-diastolic
diameter and left ventricular end-systolic diameter (D) were measured. The left ventricular
end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated. The ejection
fraction (EF) value was found, EP=(EDV-ESV)/EDV*100%.
5.3.2 Data processing
[0076] All experimental data were expressed in ±SD. GraphPad Prism6 was used for one-way
ANOVA analysis. P<0.05 indicates a significant inter-group difference; P<0.01 indicates
an extremely significant inter-group difference.
6. Experimental Results
6.1 Echocardiographic results
[0077] Echocardiography was performed after 60 consecutive days of NRG administration. The
LVEDd, LVEDs and EF of the excipient group were 1.048 ± 0.07 cm, 0.910 ± 0.09 cm and
32.1 ± 6.6%, respectively; the LVEDd, LVEDs and EF of the NRG30µg/kg/Day group were
0.981 ± 0.08 cm, 0.794 ± 0.08 cm and 43.8 ± 8.0%, respectively. According to the data
of LVEDd and LVEDs, the LVEDd and LVEDs of the everyday NRG administration group were
significantly reduced, showing an extremely significant difference from the control
group (p<0.001). According to the data of EF value, the EF value of the NRG group
increased significantly after 60 days of continuous administration, showing an extremely
significant difference from the control group (p<0.001). Frequency-unchanged and dose-reduction
treatment was given 60 days later. Echocardiography was performed in the third week.
The LVEDd, LVEDs and EF value of the control group were 1.038 ± 0.07cm, 0.899 ± 0.10
cm and 32.4+9.5%, respectively; the LVEDd, LVEDs and EF value of the NRG/30µg/kg/Day
group were 0.981 ± 0.08cm, 0.799 ± 0.08 and 42.3 ± 11.2%. The drug was completely
withdrawn after three-week dose reduction for an observation. The LVEDd, LVEDs and
EF value of the control group were 1.065 ± 0.07cm, 0.942 ± 0.10cm and 28.3 ± 9.4%,
respectively; the LVEDd, LVEDs and EF value of the NRG/30µg/kg/Day group were 0.994
± 0.08cm, 0.826 ± 0.10cm and 39.3 ± 12.7%, respectively. Echocardiography was performed
in the ninth week after withdrawal. The LVEDd, LVEDs and EF value of the control group
were 1.137 ± 0.08 cm, 1.006 ± 0.08cm and 28.0 +5.7%, respectively; the LVEDd, LVEDs
and EF value of the NRG/30µg/kg/Day group were NRG/30µg/kg/day group was 1.104 ± 0.08
cm, 0.950 ± 0.09cm, and33.4 ± 7.6%, respectively. Nine weeks after withdrawal, there
remained a significant difference in LVEDd and LVEDs between the everyday NRG administration
group and the control group; the EF value still showed a rising trend compared with
the control group. See Table 7, 8, 9 and Figure 4 for the results.
7. Conclusions
[0078] Given a certain dose and different frequencies of administration, rhNRG exerted some
therapeutic effect on myocardial infarction in rats during continuous administration,
enhancing the cardiac function of the rats with acute myocardial infarction and improving
ventricular remodeling, and deferring aging caused by myocardial infarction. It still
has a significant improving effect on the cardiac function of the rats with myocardial
infarction a long time after withdrawal.
Table 7 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction
through 60-day subcutaneous administration (x±SD)
Time |
Dose (µg/kg) |
Group |
Number of animals |
LVEDd (cm) |
LVEDs (cm) |
EF (%) |
DAY10 |
-- |
Excipient |
17 |
0.840±0.04 |
0.683±0.05 |
43.5±6.77 |
|
30 |
NRG/Day |
15 |
0.821±0.05 |
0.637±0.06 |
50.5±4.96* |
DAY30 |
-- |
Excipient |
16 |
0.940±0.05 |
0.805±0.06 |
34.4±6.82 |
|
30 |
NRG/Day |
14 |
0.912±0.06 |
0.725±0.07* |
46.8±6.37** |
DAY50 |
-- |
Excipient |
16 |
0.995±0.06 |
0.869±0.08 |
30.9±7.75 |
|
30 |
NRG/Day |
14 |
0.934±0.07* |
0.754±0.09*** |
44.4±7.85*** |
DAY60 |
-- |
Excipient |
16 |
1.048±0.07 |
0.910±0.09 |
32.1±6.61 |
|
30 |
NRG/Day |
14 |
0.981±0.08* |
0.794±0.08*** |
43.8±8.04*** |
***: p<0.001 for the subgroups after treatment compared with the excipient group;
**: p<0.01 for the subgroups after treatment compared with the excipient group;
*: p<0.05 for the subgroups after treatment compared with the excipient group |
Table 8 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction
three weeks after dose reduction following 60-day subcutaneous administration (x±SD)
Time |
Dose (µg/kg) |
Group |
Number of animals |
LVEDd (cm) |
LVEDs (cm) |
EF (%) |
DAY81 |
-- |
Excipient |
16 |
1.038±0.07 |
0.899±0.10 |
32.4±9.51 |
|
30 |
NRG/Day |
14 |
0.981±0.07** |
0.799±0.08*** |
42.3±11.20** |
***: p<0.001 for the subgroups after treatment compared with the excipient group;
**: p<0.01 for the subgroups after treatment compared with the excipient group;
*: p<0.05 for the subgroups after treatment compared with the excipient group |
Table 9 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction
after withdrawal on day 81 of subcutaneous administration (x±SD)
Time |
Dose (µg/kg) |
Group |
Number of animals |
LVEDd (cm) |
LVEDs (cm) |
EF (%) |
DAY88 |
-- |
Excipient |
17 |
1.065±0.07 |
0.942±0.10 |
28.3±9.40 |
|
30 |
NRG/Day |
15 |
0.994±0.08** |
0.826±0.10*** |
39.3±12.71*** |
DAY102 |
-- |
Excipient |
16 |
1.072±0.09 |
0.946±0.13 |
29.0±11.41 |
|
30 |
NRG/Day |
14 |
1.031±0.07* |
0.872±0.08* |
36.6±7.69** |
DAY116 |
-- |
Excipient |
16 |
1.107±0.08 |
0.985±0.12 |
27.2±9.67 |
|
30 |
NRG/Day |
14 |
1.054±0.07 |
0.908±0.08** |
33.3±5.12* |
DAY143 |
-- |
Excipient |
14 |
1.137±0.08 |
1.006±0.08 |
28.0±5.70 |
|
30 |
NRG/Day |
14 |
1.104±0.08 |
0.950±0.09 |
33.4±7.60 |
***: p<0.001 for the subgroups after treatment compared with the excipient group;
**: p<0.01 for the subgroups after treatment compared with the excipient group;
*: p<0.05 for the subgroups after treatment compared with the excipient group |
